Download Compartment-Specific Antioxidative Defense in Arabidopsis Against

Bacteriology
Compartment-Specific Antioxidative Defense in Arabidopsis
Against Virulent and Avirulent Pseudomonas syringae
Dominik K. Großkinsky, Barbara E. Koffler, Thomas Roitsch, Romana Maier, and Bernd Zechmann
First, second, third, fourth, and fifth authors: University of Graz, Institute of Plant Sciences, Schubertstrasse 51, 8010 Graz, Austria; and
fifth author: Graz University of Technology, Institute for Electron Microscopy and Fine Structure Research, Steyrergasse 17, 8010 Graz,
Austria.
Accepted for publication 20 March 2012.
ABSTRACT
Großkinsky, D. K., Koffler, B. E., Roitsch, T., Maier, R., and Zechmann,
B. 2012. Compartment-specific antioxidative defense in Arabidopsis
against virulent and avirulent Pseudomonas syringae. Phytopathology
102:662-673.
The accumulation of reactive oxygen species (ROS) during biotic
stress is either part of a hypersensitive response of the plant or induced
directly by the pathogen. Antioxidants such as ascorbate and glutathione
counteract the accumulation of ROS and are part of the defense reaction.
The aim of the present study was to investigate the compartment-specific
importance of ascorbate and glutathione during a virulent and avirulent
Pseudomonas syringae infection in Arabidopsis thaliana. Peroxisomes
were found to be the hotspot for glutathione accumulation reaching 452%
Enhanced generation of reactive oxygen species (ROS) is
commonly observed during biotic stress situations in plants
(17,39). ROS production can be induced by plants in the form of a
hypersensitive response (HR) in order to stop the spread of the
pathogen (38). This response can lead to the activation of cell
death, peroxidase catalyzed cell wall enforcement, synthesis of
secondary metabolites such as phytoalexins, and the activation of
defense genes. Additionally, the pathogen itself can produce ROS
in order to kill the host tissue and toxic components produced by
the pathogen can also lead to the accumulation of ROS in the
plant (1,17). Enhanced internal ROS production, if not counteracted by antioxidants, can have negative effects on cell structures
and plant metabolism since ROS are capable of oxidizing membrane components and proteins, which can lead to the destruction
of membranes, proteins, RNA, and DNA, causing mutation and
eventually cell death (12,37,43). The antioxidants ascorbate and
glutathione are key antagonists of ROS (12,28,29). They protect
plants against oxidative stress by detoxifying ROS, either directly
or through the ascorbate-glutathione cycle. They also play important roles in redox signaling, the modulation of defense gene
expression and they are important for the regulation of enzymatic
activities (extensively reviewed in 12,28,29). Therefore, subcellular changes in ascorbate and glutathione contents, especially
during biotic stress situations, may provide important insights into
compartment-specific defense reactions and reflect the occurrence
of compartment-specific oxidative stress. Such information can be
used as a subcellular stress marker and can be very helpful in
clarifying the importance of the protective roles of ascorbate and
Corresponding author: B. Zechmann; E-mail address: [email protected]
http://dx.doi.org/10.1094 / PHYTO-02-12-0022-R
© 2012 The American Phytopathological Society
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PHYTOPATHOLOGY
and 258% of control levels 24 h postinoculation during the virulent and
avirulent infection, respectively. An accumulation of ascorbate could also
be observed in vacuoles during Pseudomonas syringae infection, whereas
glutathione remained absent in this cell compartment. Neither glutathione
nor ascorbate accumulated in the apoplast during pathogen infection
demonstrating an only negligible role of these antioxidants in the apoplast
during pathogen infection. Compartment-specific changes followed a recently proposed stress model with an increase of ascorbate and glutathione
in most cell compartments at the early stages of infection and a strong
drop at the later stage of infection when a strong accumulation of ROS
and symptoms occurred in the leaves. This study highlights the importance of certain cell compartments and antioxidants in general for the
protection of pathogen-induced ROS accumulation.
glutathione during biotic stress situations in plants on the cellular
level.
Changes in compartment-specific glutathione contents and their
subcellular roles are well studied during virus infections in plants
(18,50,51) but there is still a great lack of knowledge regarding
bacterial infections, which differ greatly from virus infections.
Bacteria enter the leaf through stomata or wounds and grow
intercellularly by using nutrients present inside the plant (32).
Virulent bacteria are able to suppress defense reactions of the
plants and live off nutrients and water released by the plant into
the intercellular spaces (30,32,38). Damages of the host tissue
occur rather late in this interaction through the production of toxic
compounds and nutrient deprivation by the bacteria (6,32). ROS
accumulation is much weaker, correlates with symptom development and is also found in later stages of infection and leads to the
death of the infected host tissues in the long term (1,30,32,38).
Avirulent bacteria are recognized by the plant, which leads to cell
death during HR and the successful restriction of bacterial
proliferation (38,39). ROS accumulation and the development of
chlorotic and necrotic lesions occur as part of the HR in the early
stages of infection and are controlled by the plant. On the compartment-specific level, chloroplasts are considered to be the main
production center for ROS during avirulent infections (3,14,27,
36) whereas peroxisomes seem to be involved in stress signaling
as they are capable of releasing signaling molecules including
ROS (for example H2O2 and O2–) to the cytosol where they can
trigger defense gene expression (9). As antioxidants counteract
the accumulation of ROS and are important for the protection of
organelles such as the nucleus against oxidative damage (15) their
compartment-specific distribution during biotic stress can play
important roles in sensing, signaling and activating plant defense.
Thus, the aim of this study was to investigate compartmentspecific changes in ascorbate and glutathione content and to
link these changes with the accumulation of hydrogen peroxide
(H2O2) and superoxide anions (O2–) during virulent and avirulent
Pseudomonas syringae infection in Arabidopsis thaliana Col-0
plants.
MATERIALS AND METHODS
Plant material. Plants were cultivated in growth chambers
under defined conditions. After stratification for 4 days at 4°C
seeds of A. thaliana [L.] Heynh. ecotype Columbia (Col-0) were
grown on “Naturahum” potting soil (Ostendorf Gärtnereierden
GmbH, Vechta, Germany) in growth chambers with 8/16 h
day/night photoperiod. Day and night temperatures were 22 and
18°C, respectively, the relative humidity was 60%, and the plants
were kept at 100% relative soil water content. Light intensity
varied between 110 and 140 µmol m–2 s–1.
Pathogen inoculation. Infections with the virulent hemibiotrophic bacterium P. syringae pv. tomato DC3000 and a
corresponding avirulent strain P. syringae pv. tomato DC3000
AvrRpm1 were performed according to Großkinsky et al. (16).
Bacteria were precultured in culture tubes by transferring colonies
from maximum 2-week-old culture plates to 4 ml of Luria-Bertani
(LB) medium (7) containing rifampicin at 50 mg/liter (Molekula
Limited, Gillingham, Dorset, UK) and tetracycline at 10 mg/liter
(Applichem, Darmstadt, Germany) for the avirulent strain using
sterile pipette tips. Precultures were incubated overnight in an
incubator at 28°C and 180 rpm. For main cultures, 50 ml of LB
medium containing 50 mg/liter rifampicin (and 10 mg/liter
tetracycline for the avirulent strain) were inoculated with 1 ml of
the preculture in sterile Erlenmeyer flasks. The cultures were
incubated at 28°C and 180 rpm to an optical density at 600 nm
(OD600nm) between 0.4 and 0.8 (exponential growth phase). Cultures were then transferred to 50-ml falcon-tubes and centrifuged
with 3,500 rpm at 4°C for 10 min. The bacterial pellets were
resuspended in a 10 mM MgCl2 solution and adjusted to OD600nm
of 0.2 which corresponds to 1 × 108 cfu/ml (20). For infiltration,
suspensions were diluted 1:100 with a 10 mM MgCl2 solution to a
final concentration of 1 × 106 cfu/ml. Prepared suspensions were
infiltrated from the abaxial side of A. thaliana leaves using a
needleless syringe until the whole leaf was fully infiltrated by the
solution. Infiltration was carried out once on each half of the leaf
(left and right side from the middle vein). Plants were about
8 weeks old at this stage and only one fully developed leaf of the
fourth rosette was infiltrated per plant. Control plants were
infiltrated with a 10 mM MgCl2 solution without bacteria using a
needleless syringe into the abaxial side of the leaves as described
above. Harvesting of control and P. syringae-inoculated samples
was performed at the day of inoculation (=control), 12, 24, and
48 h postinoculation (hpi). For electron microscopical investigations, samples were harvested close to the middle vein where
chlorotic symptoms were observed on all leaves at the later stages
Fig. 1. Symptom development 12 h postinoculation (hpi) (B and F), 24 hpi (C and G), and 48 hpi (D and H) with Pseudomonas syringae pv. tomato. No
symptoms were observed on the controls at the time of inoculation (A) and 48 h after injection of a 10 mM MgCl2 solution without bacteria (E). First indications
of symptoms in the form of local chlorotic and necrotic spots (black circles) could be observed on leaves inoculated with the avirulent strain 12 hpi (B). Clear
symptoms (chlorotic and necrotic lesions) could be observed 24 hpi (C) with the avirulent strain which became more severe 48 hpi (D). When leaves were
inoculated with the virulent strain no symptoms could be observed 12 hpi (F). First signs of faint chlorosis were observed at 24 hpi, which turned into general
chlorosis and local necrotic lesions at 48 hpi (H). Bars = 0.5 cm.
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663
of infection. Sampling was performed 2 h after the onset of the
light period.
Sample preparation for transmission electron microscopy
and immunogold labeling. Preparation of samples for transmission electron microscopy (TEM) and immunogold labeling of
glutathione was done with ultrathin sections on nickel grids
according to Zechmann et al. (47,48). Small samples of the leaves
close to the middle vein (about 1.5 mm2) from at least three
different plants were cut on a modeling wax plate in a drop of
2.5% paraformaldehyde and 0.5% glutardialdehyde in 0.06 M
Sørensen phosphate buffer at pH 7.2 (34). Samples were then
transferred into glass vials and fixed for 90 min at room temperature (RT) in the above-mentioned solution. After fixation
samples were rinsed in 0.06 M Sørensen phosphate buffer (pH
7.2) for four times at 15 min each and dehydrated in increasing
concentrations of acetone (50, 70, and 90%) at RT for 20 min at
each step. Subsequently, specimens were gradually infiltrated
with increasing concentrations of LR-White resin (30, 60, and
100%; London Resin Company Ltd., Berkshire, UK) mixed with
acetone (90%) for a minimum of 3 h per step. Samples were
finally embedded in pure, fresh LR-White resin and polymerized
at 50°C for 48 h in small plastic containers under anaerobic
conditions. Ultrathin sections (80 nm) were cut with a Reichert
Ultracut S ultramicrotome (Leica Microsystems, Vienna, Austria).
Immunogold labeling of glutathione and ascorbate was done
with ultrathin sections on coated nickel grids with the automated
Fig. 2. Compartment specific ascorbate labeling within leaf mesophyll cells of
Pseudomonas syringae-inoculated Arabidopsis thaliana Col-0 plants. Measurements were performed 0 (=control, C, represented by the dotted line), 12, 24,
and 48 h postinoculation with the virulent strain Pseudomonas syringae pv.
tomato DC3000 (black squares) and avirulent strain Pseudomonas syringae
pv. tomato DC3000 AvrRpm1 (black circles). Data show means with standard
errors and document the amount of gold particles bound to ascorbate per
square micrometer in the respective cell compartment. n > 20 for peroxisomes
and vacuoles and n > 60 for other cell structures. Different lowercase letters
indicate significant differences between the data points. Significant differences
were calculated with the Kruskal-Wallis test followed by post-hoc comparison
according to Conover at P < 0.05.
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PHYTOPATHOLOGY
immunogold labeling system Leica EM IGL (Leica, Microsystems). The sections were blocked for 20 min with 2% bovine
serum albumin (BSA, Sigma-Aldrich, St. Louis, MO) in phosphate buffered saline (PBS, pH 7.2) and then treated with the
primary antibody against ascorbate (anti-ascorbate rat polyclonal
immunoglobulin G [IgG]; Abcam plc, Cambridge, UK) diluted
1:300 in PBS containing 1% BSA and glutathione (anti-glutathione rabbit polyclonal IgG, Millipore Corp., Billerica, MA)
diluted 1:50 in PBS containing 1% goat serum for 2 h at RT. After
a short rinse in PBS (three times at 5 min each), samples were
incubated with a 10 nm gold-conjugated secondary antibody (goat
anti-rabbit IgG for glutathione and goat anti-rat IgG for ascorbate
labeled sections, British BioCell International, Cardiff, UK)
diluted 1:50 (for sections incubated with the glutathione antibody)
and 1:100 (for sections incubated with the ascorbate antibody) in
PBS for 90 min at RT. After a short wash in PBS (3 times 5 min)
and distilled water (two times 5 min), labeled grids were either
immediately observed in a Philips CM10 transmission electron
microscope or post-stained with uranyl-acetate (2% dissolved in
aqua bidest) for 15 s.
Micrographs of randomly photographed immunogold labeled
sections were digitized and gold particles were counted automatically using the software package Cell D with the particle
analysis tool (Olympus, Life and Material Science Europa GmbH,
Hamburg, Germany) in different visually identified and manually
traced cell structures (mitochondria, plastids, nuclei, peroxisomes,
cytosol, and vacuoles). For statistical evaluation at least four
different samples were examined. A minimum of 20 (peroxisomes
and vacuoles) to 60 (other cell structures) sectioned cell structures
of at least 15 different cells were analyzed for gold particle
density per sample. The obtained data were statistically evaluated
using Statistica (Stat-Soft Europe, Hamburg, Germany) and presented as the number of gold particles per square micrometer. For
all statistical analyses the nonparametric Kruskal-Wallis test
followed by a post-hoc comparison according to Conover was
used. P < 0.05 was regarded as significant.
Determination of hydrogen peroxide by diaminobenzidine
staining. Detection of hydrogen peroxide (H2O2) was performed
according to Weigel and Glazebrook (45) by diaminobenzidine
(DAB) staining. Therefore, leaves were detached from the plants
with a razor blade at the above described time-points and immersed over night at RT in 4 ml of an aqueous solution of 1 mg/ml
DAB (pH 3.8; Roth, Karlsruhe, Germany). To clear leaves,
chlorophyll was extracted by incubation in 70% ethanol at 37°C.
All incubation steps were performed in glass vials. After chlorophyll extraction, the samples were investigated with a stereo
microscope (Olympus SZX9) and photographs were taken with a
digital camera (Olympus E330).
Determination of superoxide anions by nitroblue tetrazolium staining. Detection of superoxide anions (O2–) was performed according to Jabs et al. (19) by nitroblue tetrazolium
(NBT) staining. Therefore, leaves of different treatments were
detached from the plants with a razor blade at the above described
time-points and vacuum-infiltrated (two times at 15 s) with
10 mM NaN3 in 10 mM potassium phosphate buffer (pH 7.8) at
RT. Next, leaves were immersed at RT for 20 min in 10 mM
potassium phosphate buffer containing 0.1% NBT (AppliChem,
Darmstadt, Germany). To clear leaves, chlorophyll was extracted
by incubation in 70% ethanol at 37°C. All incubation steps were
performed in glass vials. After chlorophyll extraction, the samples
were investigated with a stereo microscope (Olympus SZX9) and
photographs were taken with a digital camera (Olympus E330).
RESULTS
Symptom development. Symptom development was evaluated
over a time period of 48 hpi on plants inoculated with the
avirulent and the virulent strain of P. syringae pv. tomato. Control
plants which were injected with a 10 mM MgCl2 solution without
bacteria did not develop symptoms throughout the time of
investigation (Fig. 1A and E). Plants inoculated with the avirulent
strain showed first symptoms in the form of local chlorotic and
necrotic spots at 12 hpi (Fig. 1B). These symptoms became more
prominent 24 hpi when yellowing occurred on the whole leaf and
necrotic spots increased in size (Fig. 1C). Symptom development
became even more severe 48 hpi when yellowing of the leaves
progressed and an increase in number and size of necrotic spots
could be observed (Fig. 1D). Leaves inoculated with the virulent
strain showed no symptoms at 12 hpi (Fig. 1F). First signs of faint
chlorosis could be observed at 24 hpi (Fig. 1G) which correlated
somewhat with the chlorosis observed on plants inoculated with
the avirulent strain at 12 hpi. A general chlorosis and local
necrotic lesions were observed on plants inoculated with the
virulent strain at 48 hpi (Fig. 1H). In general, the development of
chlorosis and necrosis occurred earlier and was more severe on
leaves inoculated with the avirulent strain when compared with
Fig. 3. Transmission electron micrographs showing the subcellular distribution of ascorbate in Arabidopsis thaliana Col-0 leaf cells inoculated with the virulent
strain Pseudomonas syringae pv. tomato DC3000 at the day of inoculation (A), 12 h postinoculation (hpi) (B), 24 hpi (C), and 48 hpi (D). C = chloroplasts with or
without starch (St), M = mitochondria, N = nuclei, Px = peroxisomes, and V = vacuoles. Bars = 1 µm.
Vol. 102, No. 7, 2012
665
symptom development on leaves inoculated with the virulent
strain.
Ascorbate. Subcellular changes of ascorbate labeling were
investigated by TEM at 12, 24, and 48 hpi of A. thaliana Col-0
plants with a virulent and avirulent strain of P. syringae. Control
plants, which were mock-inoculated by injection of a 10 mM
MgCl2 solution without bacteria, showed similar (not significantly
different) subcellular ascorbate contents 12, 24, and 48 h after the
injection of the solution (data not shown). Therefore, a mean of
the determined subcellular ascorbate contents of these time-points
was calculated, defined as control and used for calculating
significant differences between mock and P. syringae-inoculated
plants at 12, 24, and 48 hpi. Subcellular ascorbate contents significantly increased (75%) 12 hpi in mitochondria during the
avirulent infection but remained statistically unchanged in the
compatible interaction (Figs. 2 to 4). Unchanged levels of ascorbate were found in mitochondria during the virulent and avirulent
infection 24 hpi. Whereas mitochondria showed a significant
Fig. 4. Transmission electron micrographs showing the subcellular distribution of ascorbate in Arabidopsis thaliana Col-0 leaf cells inoculated with the avirulent
strain Pseudomonas syringae pv. tomato DC3000 AvrRpm1 at the day of inoculation (A), 12 h postinoculation (hpi) (B), 24 hpi (C), and 48 hpi (D). C =
chloroplasts with or without starch (St), CW = cell walls, M = mitochondria, N = nuclei, Px = peroxisomes, and V = vacuoles. Bars = 1 and 0.5 µm in inset.
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PHYTOPATHOLOGY
decrease of ascorbate contents of about 29% during the avirulent
infection, unchanged levels were detected during the virulent
infection 48 hpi. A significant increase of ascorbate contents was
observed in chloroplasts during the avirulent (36% at 12 hpi, 54%
at 24 hpi, and 25% at 48 hpi) and virulent (110% at 12 hpi, 46%
at 24 hpi, and 28% at 48 hpi) infection over the whole time period
(Fig. 2). Ascorbate contents in nuclei were significantly increased
(109%) 12 hpi during the virulent infection but remained statistically unchanged in the incompatible interaction. A decrease
(37%) of ascorbate specific labeling was observed 24 hpi in the
virulent infection whereas an increase of about 77% was found at
this time-point in the avirulent infection. Decreased ascorbate
contents of 56 and 78% in comparison to the control were observed in nuclei 48 hpi during the virulent and avirulent infection
(Fig. 2). In peroxisomes unchanged levels of ascorbate were
detected 12 hpi during the virulent infection, whereas significant
decreased levels of ascorbate specific labeling (70%) were detected during the avirulent infection at this time-point. A decrease
of ascorbate labeling in peroxisomes was observed at 24 hpi and
48 hpi in the virulent (30 and 69%) and the avirulent infection (56
and 57%). Ascorbate accumulated in the cytosol at 12 hpi during
the virulent (113%) and the avirulent (23%) infection (Fig. 2).
Whereas a significant decrease in ascorbate specific labeling was
observed 24 hpi in the virulent infection (29%), similar levels as
observed in the control occurred in the cytosol during the
avirulent infection. A decrease of ascorbate labeling was found
during the virulent (56%) and the avirulent (54%) infection 48
hpi. An accumulation of ascorbate specific labeling was detected
in vacuoles in the virulent (103% at 12 hpi, 88% at 24 hpi, and
84% at 48 hpi) and the avirulent (111% at 12 hpi, 74% at 24 hpi,
and 32% at 48 hpi) infection (Figs. 2 to 4). Ascorbate could not be
detected in the apoplast of control plants and plants infected with
the virulent and avirulent P. syringae strain (Figs. 3 and 4).
Glutathione. Subcellular changes of glutathione labeling were
investigated by TEM at 12, 24, and 48 hpi of A. thaliana Col-0
plants with a virulent and avirulent strain of P. syringae. Control
plants, which were mock-inoculated by injection of a 10 mM
MgCl2 solution without bacteria, showed similar (not significantly
different) subcellular glutathione contents at 12, 24, and 48 h after
the injection of the solution (data not shown). Therefore, a mean
of the determined subcellular glutathione contents of these timepoints was calculated, defined as control and used for calculating
significant differences between mock and P. syringae inoculated
plants at 12, 24, and 48 hpi. Subcellular glutathione contents were
heavily affected by P. syringae infection. In mitochondria, an
initial significant increase of glutathione of 44 and 29% was
detected 12 hpi during the virulent and the avirulent infection
(Figs. 5 to 7). Glutathione contents remained statistically unchanged 24 hpi and significantly decreased about 29% in the
virulent infection 48 hpi. In chloroplasts, a significant increase of
glutathione labeling was detected in the virulent (73%) and avirulent (25%) infection 12 hpi. Whereas glutathione contents were
even more strongly increased in the virulent infection 24 hpi
(133%), control values were detected in the chloroplasts in the
avirulent infection at this stage (Fig. 5). Glutathione contents
were significantly decreased in chloroplasts 48 hpi during the
virulent (48%) and avirulent (40%) infection. Glutathione contents in nuclei were statistically elevated to about 53% in the
avirulent infection 12 hpi but remained at control values in the
virulent interaction. A significant increase in nuclei of about 158
and 70% was detected 24 hpi during the virulent and avirulent
infection, respectively (Fig. 5). At 48 hpi, glutathione contents
were significantly increased to 70 and 38% in nuclei during the
compatible and incompatible interaction. In peroxisomes glutathione contents remained similar to control values in the virulent
infection but were significantly increased (139%) during the
avirulent infection 12 hpi (Fig. 5). A strong increase of glutathione-specific labeling during the virulent and the avirulent
infection (452 and 258%) was found 24 hpi. At 48 hpi, a strong
decrease of glutathione contents of about 48 and 39% was found
during the compatible and incompatible interaction. Glutathione
contents in the cytosol were significantly increased 12 hpi during
the avirulent infection (143%) but showed unchanged levels
during the virulent infection (Fig. 5). A strong increase of glutathione contents of 307 and 160% was found 24 hpi in the virulent
and avirulent infection, respectively. In the virulent infection
glutathione labeling remained unchanged at 48 hpi but was
significantly decreased during the avirulent infection (41%) (Figs.
5 to 7). Glutathione could not be detected in the apoplast and
vacuoles of control plants and plants infected with the virulent
and avirulent P. syringae strain (Figs. 6 and 7).
Determination of hydrogen peroxide by DAB staining. DAB
staining revealed the first signs of H2O2 accumulation in the form
of small light brown stained areas on the leaves of plants
inoculated with the avirulent strain of P. syringae 12 hpi (Fig.
8B). At this time-point no staining was observed on plants
inoculated with the virulent strain (Fig. 8F). H2O2 accumulation,
visualized in the form of larger light brown stained areas on the
leaves, became more prominent at 24 hpi and reached its maximum at 48 hpi on leaves of plants inoculated with the avirulent
strain when most of the leaf was heavily stained by DAB (Fig. 8C
and D, respectively). The first signs of H2O2 accumulation in the
form of brown spots became visible on plants inoculated with the
virulent strain at 24 hpi and reached its maximum at 48 hpi when
Fig. 5. Compartment specific glutathione labeling within leaf mesophyll cells
of Pseudomonas syringae-inoculated Arabidopsis thaliana Col-0 plants.
Measurements were performed 0 (=control, C, represented by the dotted line),
12, 24, and 48 h postinoculation with the virulent strain Pseudomonas
syringae pv. tomato DC3000 (black squares) and avirulent strain Pseudomonas syringae pv. tomato DC3000 AvrRpm1 (black circles). Data show
means with standard errors and document the amount of gold particles bound
to glutathione per square micrometer in the respective cell compartment. n >
20 for peroxisomes and vacuoles and n > 60 for other cell structures. Different
lowercase letters indicate significant differences between the data points.
Significant differences were calculated with the Kruskal-Wallis test followed
by post-hoc comparison according to Conover at P < 0.05.
Vol. 102, No. 7, 2012
667
larger light brown areas appeared on the leaves (Fig. 8G and H).
Staining by far did not reach the same intensity as found in leaves
inoculated with the avirulent strain at 48 hpi. No or only very
little DAB staining could be observed on control plants at 0 h and
48 hpi (Fig. 8A and E, respectively).
Determination of superoxide anions by NBT staining. A
strong accumulation of O2– could be observed in the form of blue
stained areas all over the leaves of plants inoculated with the
avirulent strain of P. syringae at 12 hpi (Fig. 9B). NBT staining
remained similar during the avirulent infection 24 hpi and 48 hpi
(Fig. 9B to D). The accumulation of O2– was much weaker in
plants inoculated with the virulent strain where first signs of O2–
could be observed 24 hpi. Only small blue stained areas indicating the presence of O2– could be observed at this time (Fig.
9G). A strong accumulation of O2– could be observed 48 hpi in
leaves of plants inoculated with the virulent strain of P. syringae
Fig. 6. Transmission electron micrographs showing the subcellular distribution of glutathione in Arabidopsis thaliana Col-0 leaf cells inoculated with the virulent
strain Pseudomonas syringae pv. tomato DC3000 at the day of inoculation (A), 12 h postinoculation (hpi) (B), 24 h (C), and 48 h (D) after inoculation. C =
chloroplasts with or without starch (St), IS = intercellular space, M = mitochondria, N = nuclei, Px = peroxisomes, and V = vacuoles. Bars = 1 and 0.5 µm in
insets.
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PHYTOPATHOLOGY
(Fig. 9H). No or only very little NBT staining could be observed
on control plants at 0 h and 48 hpi (Fig. 9A and E).
DISCUSSION
The results revealed clear differences in the accumulation of
ROS and the counteracting antioxidants ascorbate and glutathione
between the virulent and avirulent P. syringae infection in A.
thaliana. Plants infected with the avirulent strain showed a much
earlier and stronger accumulation of H2O2 and O2– (starting 12 hpi
with heaviest accumulation 48 hpi) than the virulent strain where
first signs could only be observed at 24 hpi (Figs. 8 and 9).
Avirulent infections are characterized by the induction of HR,
which leads to the induction of cell death mediated by the
accumulation of ROS early in the infection period as observed in
this study in order to constrain the spread of the pathogen (38).
Fig. 7. Transmission electron micrographs showing the subcellular distribution of glutathione in Arabidopsis thaliana Col-0 leaf cells inoculated with the avirulent
strain Pseudomonas syringae pv. tomato DC3000 AvrRpm1 at the day of inoculation (A), 12 h postinoculation (hpi) (B), 24 hpi (C), and 48 hpi (D). C =
chloroplasts with or without starch (St), CW = cell walls, IS = intercellular space, M = mitochondria, N = nuclei, Px = peroxisomes, and V = vacuoles. Bars = 1
and 0.5 µm in insets.
Vol. 102, No. 7, 2012
669
The accumulation of ROS starting at 12 hpi during the avirulent
infection was accompanied by a strong accumulation of ascorbate
and glutathione in most cell compartments during the early stages
of infection (12 and 24 hpi) and a sharp drop at 48 hpi when ROS
reached their maximum levels (Figs. 8 and 9). These results
clearly demonstrate that ascorbate and glutathione counteracted
the accumulation of ROS during the early stages of the HR but
were not able to keep the accumulation of ROS under control in
the long term. ROS accumulation was eventually leading to the
development of chlorotic and necrotic lesions, which are typical
signs observed during HR (Fig. 1). A different effect was found
during the virulent infection. Whereas ascorbate contents showed
the strongest increase 12 hpi (Fig. 2), glutathione contents
accumulated later at 24 hpi (Fig. 5) when first signs of ROS
accumulation could be observed by light microscopy during the
virulent infection (Figs. 8 and 9). Compatible interactions are
characterized by the induction of ROS in the later stages of
infection either by the bacteria itself or by toxic components
released by the bacteria inside the infected tissue (6,17,30,32,38).
Thus, we can conclude that the accumulation of glutathione
contents correlated well with the occurrence of oxidative stress
(12 and 24 hpi in the avirulent and virulent infection, respectively) highlighting the importance of glutathione in the protection of P. syringae-infected A. thaliana plants against ROS (1). As
a strong accumulation of ascorbate was additionally found prior
to the occurrence of ROS (12 hpi) in the virulent infection (Fig.
2), it can also be concluded that ascorbate might take over
additional roles in the defense response (e.g., signaling of basal
resistance) besides solely the detoxification of ROS. Even though
glutathione accumulated later (24 hpi) during the virulent infection (Fig. 5), it can be summarized that in general the virulent and
avirulent P. syringae infection are both characterized by an
accumulation of ascorbate and glutathione in most cell compartments in the early stages of infection and by a strong drop at
48 hpi when ROS accumulation is strongest and symptom development in the form of chlorotic and necrotic lesions appear
(Fig. 1). These results fit well in a recently proposed general
stress model for plant material involving antioxidants (22). In the
proposed model, plants activate the antioxidant system during the
alarming and resistance phase, which correlates well with the
accumulation of glutathione and ascorbate during the early
stages of infection (12 and 24 hpi) in this study. Antioxidants
play important roles in these stages for stress signaling, protection
and repair of plant compounds and metabolic pathways.
The exhaustion phase is defined by the increasing failure of
protection and repair mechanisms correlating with decreased
concentrations of antioxidants and accumulation of ROS, which
eventually lead to cell death (22), as could be observed during the
later stages of P. syringae infection in A. thaliana in this study
(Fig. 1).
Fig. 8. Visualization of hydrogen peroxide (H2O2) by diaminobenzidine (DAB)-staining 12 h postinoculation (hpi) (B and F), 24 hpi (C and G), and 48 hpi (D
and H) with Pseudomonas syringae pv. tomato. No staining was observed on the controls at the time of inoculation (A) and 48 h after injection of a 10 mM
MgCl2 solution without bacteria (E). First H2O2 staining in the form of small dark spots (arrows) was observed 12 hpi (B) in the avirulent infection, which got
larger (circles) 24 hpi (C) and reached its maximum expansion at 48 hpi when large areas of the leaves could be stained (D). No staining could be observed 12 hpi
on leaves inoculated with the virulent strain (F). First small spots of H2O2 accumulation (arrows) were observed 24 hpi (G), which increased in size (circles) 48
hpi (H) on plants inoculated with the virulent strain of Pseudomonas syringae pv. tomato. Bars = 0.5 cm.
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PHYTOPATHOLOGY
On the subcellular level it is interesting that peroxisomes reacted with the strongest increase in glutathione content of all cell
compartments at the early stages of the avirulent and virulent
infection (12 and 24 hpi; Fig. 5 provides details). At the end of the
infection period (48 hpi) both ascorbate and glutathione were
strongly decreased compared with the control (Figs. 2 and 5)
which correlated with a strong accumulation of H2O2 and O2–
(Figs. 8 and 9) and the development of chlorotic and necrotic
symptoms (Fig. 1). Similar results have also been obtained for
peroxisomes during Botrytis cinerea infection in tomato where
the initial increase of the antioxidative system in peroxisomes
before the development of symptoms was followed by a strong
decrease, which correlated with the appearance of infection
symptoms (23,24). The authors concluded that the initial increase
of the antioxidative system was due to the necessity to detoxify
H2O2, whereas the collapse of the antioxidative system in peroxisomes at the later stages promoted leaf senescence that favored
diseases development. Recently, peroxisomes have also been
described as subcellular indicators and sensors of plant stress
since they are capable of releasing signaling molecules including
ROS (for example H2O2 and O2–) into the cytosol where they can
trigger defense gene expression, which can lead to stress
signaling and the activation of plant defense (9). As ROS are also
key messenger molecules for signaling of pathogen-induced cell
death, the observed collapse of the antioxidative system in peroxisomes (Figs. 2 and 5) and the correlating accumulation of ROS
(Figs. 8 and 9) at the later stages could be important triggers for
the progress of diseases symptoms as observed during bacterial
and fungal diseases (9,23,24,38).
Ascorbate and glutathione contents in the cytosol and nuclei
reacted similarly to the pathogen induced oxidative stress. An
initial increase of ascorbate and glutathione contents (12 and 24
hpi) during the avirulent infection was followed by a drop of both
antioxidants at the late stage of infection (48 hpi; Figs. 2 and 5
provide details), which correlated with ROS accumulation (Figs.
8 and 9) and symptom development (Fig. 1). Even though the
exact roles of ascorbate and glutathione in nuclei during pathogen
attack are not completely clear, their accumulation in the early
stages of infection highlights the important role of these
antioxidants in the protection of the nucleus. It has been recently
addressed that antioxidants such as glutathione fulfill pivotal roles
in nuclear function in plant and animal tissue since it is important
for DNA synthesis, cell proliferation, and regulation of the
nuclear matrix organization and proteins (10,13). Additionally, it
has been observed that the reduced form of glutathione in the
nucleus protects DNA from oxidative damage, as the latter were
negatively correlated with high reduced glutathione contents in
the nucleus (15). Therefore, it could be possible that the strong
elevation of ascorbate and glutathione observed in nuclei at the
early stages of P. syringae infection (Figs. 2 and 5) could be
important for maintaining and preserving function of nuclei
during events of oxidative stress.
Fig. 9. Visualization of superoxide anions (O2–) by nitroblue tetrazolium (NBT)-staining 12 h postinoculation (hpi) (B and F), 24 hpi (C and G), and 48 hpi (D
and H) with Pseudomonas syringae pv. tomato. No staining was observed on the controls at the time of inoculation (A) and 48 h after injection of a 10 mM
MgCl2 solution without bacteria (E). First O2– staining in form of large stained areas was observed 12 hpi (B) in the avirulent infection and remained similar at 24
and 48 hpi (C and D). No staining could be observed 12 hpi on leaves inoculated with the virulent strain (F). First small spots of O2– accumulation (circles) were
observed 24 hpi (G), which increased in size 48 hpi (H) on plants inoculated with the virulent strain of Pseudomonas syringae pv. tomato. Bars = 0.5cm.
Vol. 102, No. 7, 2012
671
It is interesting that chloroplasts showed a strong accumulation
of ascorbate over the whole investigation period in the virulent
and avirulent infection (Fig. 2). Chloroplasts are considered to be
the main production center for ROS during avirulent infections
(3,14,27,36). As ascorbate in A. thaliana is solely produced in
mitochondria through the L-galactose pathway (5,11,26,46), these
results indicate that chloroplasts accumulate ascorbate through
import from the cytosol despite a strong drop of ascorbate in most
cell compartments starting at 24 hpi (Fig. 2). This observation is
even more impressive considering that glutathione contents were
significantly decreased 48 hpi in chloroplasts (Fig. 5), which indicates that at the later stages of infection ascorbate contents mainly
take over the antioxidative protection of the chloroplasts.
A strong accumulation of ascorbate was observed over the
whole period of investigation in vacuoles during the avirulent and
virulent infection (Figs. 2 to 4). These results are interesting in
context to the observed accumulation of ascorbate in vacuoles
during other stress situations such as high light stress (49). It has
been proposed recently that ascorbate is involved in the detoxification of H2O2, which diffuses into vacuoles during situations of
oxidative stress (35), such as observed during P. syringae infection in this study. In vacuoles, ascorbate helps to reduce phenoxyl
radicals, created by oxidation of phenols by H2O2, and is oxidized
to mono- and dehydroascorbic acid, which is then transported into
the cytosol for reduction to ascorbic acid (35). Therefore, the
results of this study are another important indication of possible
protective roles of ascorbate in vacuoles against oxidative stress.
Ascorbate and glutathione could not be detected in the apoplast
of control and P. syringae-infected plants (Figs. 3A, 4A, 6A, and
7A). This is surprising as ascorbate in the apoplast is considered
to be the only significant redox buffer (12,31) and to play an
important role in the protection against ozone (4,8,33). Similar to
ozone stress, P. syringae infection also causes the induction of
ROS primary in the apoplast after entering the leaves through
stomata and wounds and growing intercellularly in the infected
tissue (32,39). Thus, an accumulation of ascorbate (and glutathione) could have been expected also in the apoplast of P.
syringae-infected plant tissue. The lack of labeling could either be
explained by the inability of the present antibody approach to
visualize ascorbate and glutathione in the apoplast or to the lack
of an accumulation of antioxidants in the apoplast during P.
syringae infection. In other plant species, antioxidants such as
ascorbate and glutathione were found to occur in quite low
amounts in the apoplast in comparison to foliar levels in nonstressed plants (21,40,41,42,44) or could not be detected at all
(25) even under situations of oxidative stress such as excessive
light exposure (49). Additionally, a decrease in ascorbate and
glutathione contents measured with biochemical methods in the
apoplast has been observed in several plant species during pathogen attack (40,41,42). Thus, the lack of labeling in the apoplast
found in this study (Figs. 3A, 4A, 6A, and 7A) most likely
indicates that ascorbate and glutathione in the apoplast play only
a minor role during for the protection against ROS induced by P.
syringae infection in A. thaliana plants.
Summing up, the study demonstrated that the occurrence of
oxidative stress (12 and 24 hpi in the avirulent and virulent
infection, respectively) correlated well with an accumulation of
glutathione in most cell compartments, indicating the importance
of glutathione in the protection against oxidative stress and basal
plant resistance. A strong accumulation of ascorbate was
additionally found prior to the occurrence of ROS (12 hpi) in the
virulent infection indicating that ascorbate might take over additional roles in the defense response (e.g., signaling of basal resistance) besides solely the detoxification of ROS. Despite these
differences, ascorbate and glutathione contents followed a general
stress model in most cell compartments. An increase in ascorbate
and glutathione contents at the early stages of infection emphasizes the importance of these antioxidants for stress signaling,
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PHYTOPATHOLOGY
protection and repair of plant compounds and metabolic pathways
during the alarming and resistance phase. The strong drop of
ascorbate and glutathione at the later stages of infection indicate
the failure of protection and repair mechanisms, which correlated
with ROS accumulation and symptom development such as the
induction of chlorotic and necrotic lesions. On the subcellular
level, peroxisomes could be defined as the hotspots of glutathione
accumulation, which highlights the importance of peroxisomes in
plant stress responses to pathogen attack.
ACKNOWLEDGMENTS
This work was supported by the Austrian Science Fund (FWF, P20619
and P22988 to B. Zechmann).
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